G protein coupled receptors (GPCRs) play critical roles in biologically and medically important signaling; however, the molecular mechanisms of activation of intracellular G proteins by ligand-bound GPCRs are not understood. The yeast pheromone response pathway provides an example of GPCR signaling that can be genetically manipulated in ways that would be impossible in other types of cells, yet components of this pathway are functionally interchangeable with corresponding components of mammalian systems. This project will use genetic approaches only possible in yeast to pursue a step-by-step dissection of the mechanisms of GPCR signaling by heterologously expressed somatostatin type 2 receptor and the endogenous yeast receptor for the a-mating pheromone, two receptors that can activate the same G protein but share no sequence similarity. The aims of the project are: I. Identification of sites of G protein interaction with receptors. To address the question of how activation of a GPCR leads to nucleotide exchange by a G protein and to map the interfaces between the two types of receptors and G proteins, a genetic screen will be conducted to identify mutations in G protein subunits that specifically prevent the interaction with receptors and mutations in receptors that specifically compensate for defects in G protein subunits. II. Identification of the conformational changes that constitute receptor activation. A new technique for pair-wise random introduction of cysteine residues will be used to screen for mutant receptor alleles that are locked in the activated state by disulfide bonds as a way of identifying sites that undergo intramolecular motion upon activation of receptors. III. Identification of ligand-receptor interactions mediating responses to different types of ligands. Screens will be conducted to identify mutant somatostatin receptors with altered responses to certain ligands and altered somatostatin ligands with altered effects on signaling. The goal is to identify particular sites of ligand-receptor interaction that are specific for activated and un-activated states of the receptor. IV. Determination of effects of receptor oligomerization on signaling and identification of sites of receptor-receptor interaction. A new flow cytometry-based assay will be used to screen libraries of randomly mutagenized receptors to identify amino acid substitutions that specifically block receptor oligomerization. The mutant receptors will then be used to determine the effects of oligomerization on signaling function.